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Xenobiotica
the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
The metabolism and drug-drug interaction
potential of the selective prostacyclin receptor
agonist selexipag
Carmela Gnerre, Jérôme Segrestaa, Swen Seeland, Päivi Äänismaa, Thomas
Pfeifer, Stephane Delahaye, Ruben de Kanter, Tomohiko Ichikawa, Tetsuhiro
Yamada & Alexander Treiber
To cite this article: Carmela Gnerre, Jérôme Segrestaa, Swen Seeland, Päivi Äänismaa, Thomas
Pfeifer, Stephane Delahaye, Ruben de Kanter, Tomohiko Ichikawa, Tetsuhiro Yamada & Alexander
Treiber (2017): The metabolism and drug-drug interaction potential of the selective prostacyclin
receptor agonist selexipag, Xenobiotica, DOI: 10.1080/00498254.2017.1357088
To link to this article: http://dx.doi.org/10.1080/00498254.2017.1357088
Accepted author version posted online: 24
Jul 2017.
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The metabolism and drug-drug interaction potential of the selective
prostacyclin receptor agonist selexipag
Carmela Gnerre
1
, Jérôme Segrestaa
1
, Swen Seeland
1
, Päivi Äänismaa
1
, Thomas Pfeifer
1*
,
Stephane Delahaye
1
, Ruben de Kanter
1
, Tomohiko Ichikawa
2
, Tetsuhiro Yamada
2
and
Alexander Treiber
1
.
1
Preclinical Pharmacokinetics and Metabolism, Actelion Pharmaceuticals Ltd, Allschwil, and
2
Pharmacokinetics and Safety Assessment Dept., Nippon Shinyaku Co., Ltd, Kyoto, Japan
*
Current address: Märtktweg 30, D-79576 Weil am Rhein
Corresponding Author: Carmela Gnerre, Idorsia Pharmaceuticals Ltd, Hegenheimermattweg 91, CH-4123
Allschwil, Switzerland. Email: carmela.gnerre@idorsia.com
Acknowledgments
The authors would like to thank Noura Akel, Julia Friedrich, Joëlle Pichot, Ervan Hauy and
Ali Selimi for their dedication and experimental contributions and Hashem Salloukh and
Alexey Veligodskiy for their assistance in the preparation of this manuscript.
Declaration of interest
This work was funded by Actelion Pharmaceuticals Ltd, Allschwil, Switzerland and Nippon
Shinyaku Co, Ltd, Kyoto, Japan. The authors report no declarations of interest.
All experiments described in this report have been conducted in the research facilities of
Actelion Pharmaceuticals Ltd and Nippon Shinyaku. During manuscript review, Actelion
Pharmaceuticals Ltd was acquired by Johnson & Johnson, and its drug discovery and early
development activities subsequently transferred into a newly created company, Idorsia
Pharmaceuticals Ltd. With the exception of Thomas Pfeifer, Tetsuhiro Yamada and
Tomohiko Ichikawa, all authors of this report were employees of Idorsia Pharmaceuticals Ltd
at the time of manuscript publication.
Accepted Manuscript
The metabolism and drug-drug interaction potential of the selective
prostacyclin receptor agonist selexipag
Abstract
- The metabolism of selexipag has been studied in vivo in man and the main excreted
metabolites were identified. Also, metabolites circulating in human plasma have been
structurally identified and quantified.
- The main metabolic pathway of selexipag in man is the formation of the active metabolite
ACT-333679. Other metabolic pathways include oxidation and dealkylation reactions. All
primary metabolites undergo subsequent hydrolysis of the sulphonamide moiety to their
corresponding acids. ACT-333679 undergoes conjugation with glucuronic acid and aromatic
hydroxylation to P10, the main metabolite detected in human faeces.
- The formation of the active metabolite ACT-333679 is catalysed by carboxylesterases,
while the oxidation and dealkylation reactions are metabolized by CYP2C8 and CYP3A4.
CYP2C8 is the only P450 isoform catalyzing the aromatic hydroxylation to P10. CYP2C8
together with CYP3A4 are also involved in the formation of several minor ACT-333679
metabolites. UGT1A3 and UGT2B7 catalyze the glucuronidation of ACT-333679.
- The potential of selexipag to inhibit or induce cytochrome P450 enzymes or drug transport
proteins was studied in vitro. Selexipag is an inhibitor of CYP2C8 and CYP2C9 and induces
CYP3A4 and CYP2C9 in vitro. Also, selexipag inhibits the transporters OATP1B1,
OATP1B3, OAT1, OAT3 and BCRP. However, due to its low dose and relatively low
unbound exposure, selexipag has a low potential for causing drug-drug interactions.
Accepted Manuscript
Word Count:
Abstract (213), Introduction (506), Materials and Methods (5238), Results (1761) Discussion
(1057), References (722), Table legends (291) Total (9788)
Key words: Prostacyclin receptor agonist, selexipag, metabolism in man, drug
interaction, cytochrome P450, drug transporter
Accepted Manuscript
Introduction
Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by
endothelial dysfunction, impaired signalling and vascular remodelling, leading to right
ventricular dysfunction and eventually failure if left untreated (Galiè et al., 2015; Simoneau
et al., 2013). In PAH, there is increased production of vasoconstrictor mediators such as
endothelin, and reduced production of the vasodilator mediators prostacyclin and nitric oxide
(Clapp and Gurung, 2015; Tuder et al., 1999). Approved PAH-specific pulmonary vasoactive
therapies target three major pathophysiological pathways, i.e. the nitric oxide, endothelin-1
and prostacyclin pathways. They comprise a soluble guanylate cyclase stimulator,
phosphodiesterase-5 inhibitors, endothelin receptor antagonists and prostanoids analogues
(Humbert and Ghofrani, 2016; Gailè et al., 2013; Taichman et al., 2014). Selexipag is an
orally active, non-prostanoid, selective prostacyclin receptor agonist, which is rapidly
hydrolysed to its active metabolite ACT-333679. Selexipag (Uptravi
®
) was approved recently
in several countries/regions including the USA and Europe based on results of the large,
randomized, placebo-controlled, event-driven clinical study GRIPHON. In that study,
selexipag when given to PAH patients significantly reduced the risk of the primary composite
endpoint of all-cause death or a complication related to PAH versus placebo (Sardana et al.,
2016; Duggan et al., 2017).
The approved treatment of selexipag is based on an individual uptitration scheme. The
starting dose is 200 μg twice daily, with increases of 200 μg twice daily every week up to the
highest tolerated dose of 1600 μg twice daily (Hardin and Chin, 2016). Selexipag is rapidly
absorbed, with maximum plasma concentration achieved within 2 hours post-dose. Plasma
exposure to ACT-333679 is approximately 4 times higher than that to selexipag (Bruderer et
al., 2014). The oral bioavailability of selexipag is 49% (Kaufmann et al., 2017). It has been
demonstrated that ACT-333679 is a 37-fold more potent, selective agonist at human IP
Accepted Manuscript
receptors than selexipag (Gatfield et al., 2017). This potency difference, together with the
higher unbound abundance of ACT-333679 in plasma as compared to selexipag, makes it the
most relevant pharmacological entity.
Selexipag and ACT-333679 are lipophilic molecules with logD values of 2.1 and 2.2,
respectively. Both compounds are negatively charged at physiological pH and highly bound
to plasma proteins.
Here, we describe the metabolism and the drug-drug interaction potential of selexipag in
man. Radiolabelled selexipag was given orally to healthy volunteers, and plasma and excreta
collected for up to 7 days. The chemical structures of the most prominent faecal metabolites
were determined but metabolite structures could not be identified in plasma and urine due to
the low absolute radioactivity concentrations. To further understand selexipag's metabolic
pathways, plasma samples from a clinical phase I study (Bruderer et al., 2014) were used to
determine the chemical nature and quantity of circulating metabolites. Also, metabolism of
radiolabelled selexipag and ACT-333679 was studied in vitro with human plasma and liver
preparations. Metabolic patterns were recorded using high performance liquid
chromatography (HPLC) coupled to radiodetection and metabolites structures were identified
using mass spectrometry (MS) technology and authentic references. The potential of both
compounds to inhibit or induce cytochrome P450 enzymes or drug transporters was
investigated in vitro.
Accepted Manuscript
Material and Methods
Chemicals and Reagents
[
14
C]-Selexipag (ACT-293987) and its metabolite [
14
C]-ACT-333679 (Figure 1) were
manufactured at Nippon Shinyaku (Kyoto, Japan). The specific activities of [
14
C]-selexipag
and [
14
C]-ACT-333679 were 56.2 and 56.9 mCi/mmol, respectively. The radiochemical
purity of both compounds was 98% or above. Non-labelled selexipag (lot 32 or 25) and ACT-
333679 (lot 10) were manufactured at Nippon Shinyaku with a purity of 99.7 % or higher.
The deuterated standards ACT-293987B (batch 2122-DB/70) and ACT-333679B (batch
2122-AA/60) used for bioanalytical purposes were synthesized at Actelion Pharmaceuticals
Ltd (Allschwil, Switzerland). Metabolites P4, P10, P11, P12, P13, P14, P18, P34 and P35
were synthesized at Nippon Shinyaku. All other chemicals and solvents used throughout this
study were of highest commercially available quality and obtained from Sigma-Aldrich,
Fluka (Buchs, Switzerland), American Radiolabeled Chemicals (St. Louis, USA), NEN
Radiochemicals Perkin Elmer (Boston, USA), Nacalai Tesque (Kyoto, Japan) or Wako
Chemicals (Osaka, Japan). Liquid scintillation cocktails, Emulsifier Scintillator Plus, Hionic-
Fluor Irga Safe Plus and Optiflow Safe 2, were from Perkin Elmer (Zürich, Switzerland or
Waltham, USA) and Berthold Technologies (Regensdorf, Switzerland). Recombinant
cytochrome P450 and UGT enzymes were purchased from Cypex Ltd (Dundee, United
Kingdom) or BD Biosciences (Japan). Pooled human liver microsomes were obtained from
BD Biosciences (Basel, Switzerland or Tokyo, Japan), Xenotech (Kansas City, USA) or
Biopredic International (Rennes, France), and fresh human hepatocytes were obtained from
Cytonet (Hannover, Germany).
Accepted Manuscript
Identification of metabolites in human faeces
Oral dosing with [
14
C]-selexipag in human and preparation of faecal extracts
A single-centre, open-label, non-randomized study after single dose administration of 400 µg
selexipag and a radioactive dose of 45 µCi was carried out in 6 subjects (Kaufmann et al.,
2012). This study was conducted in compliance with the principles of the Declaration of
Helsinki. The study protocol was approved by the Edinburgh Independent Ethics Committee
for Medical Research. The faeces collected from that study at time intervals of 24 h up to 168
h post-dose were homogenized in an approximately equal weight of water/acetone. Faecal
homogenate samples from each time interval were twice centrifuged at 50 g and metabolites
were extracted after acidification with 1M hydrochloric acid with ethyl acetate. After mixing
for 10 minutes, the samples were further centrifuged at 2160 g for 10 min.
Radioactive metabolite profiling
Radioactivity profiling was performed using an Agilent 1100 HPLC system with an L-
column ODS (5 μm; 150 × 4.6 mm ID; Chemicals Evaluation and Research Institute, Tokyo,
Japan) coupled in-line with a PerkinElmer D-515TR radiodetector. The mobile phase was
acetonitrile/10 mM ammonium acetate, pH 4.95 (10:90, v/v) as phase A and
acetonitrile/10 mM ammonium acetate, pH 4.95 (90:10, v/v) as phase B. The HPLC flow rate
was 1 mL/min and the column temperature was 40°C. The radiodetector utilized a 500-μL
liquid cell with a scintillant (Ultima Flo AP) flow rate of 2 mL/min. The drug and metabolites
were eluted with the following gradient program: starting from 20% B to 70% B at 30 min,
then to 100% B at 30.01 min and maintained until 33.01 min; then back to 20% B at 33.01
min. Radiochromatograms generated from the radiometry were processed using ChemStation
(Agilent) and the Flo-One software package (version 3.6, PerkinElmer).
Accepted Manuscript
Metabolite identification
The structures of main metabolites in human faecal samples were elucidated using
LC/radiodetection and mass spectrometry in comparison with synthetic standards. In
addition, the results of incubation of [
14
C]ACT-333679 with recombinant CYP2C8 (Nosan
Corporation (Kanagawa, Japan) were used to support the identification and structural
elucidation of those metabolites. The incubation mixture contained 75 μg/mL CYP2C8
recombinant protein, 20 μg/mL [
14
C]ACT-333679, 2.7 μCi/mL, 1 mM NADPH, 3.3 mM
MgCl
2
, and 100 mM potassium phosphate buffer (pH 7.4). Non-labelled ACT-333679 was
incubated using the same procedure. Reactions were initiated by the addition of NADPH and
incubated at 37 °C. After 120 min, reactions were terminated with 2.5 mL of methanol. The
mixtures were centrifuged at 2160 g for 10 min at room temperature and the supernatants
were subjected to HPLC analysis. For LC/radiochromatography, an Agilent 1100 HPLC
system coupled in-line with a 515TR radiodetector was used. To isolate a peak with a
retention time identical to that of the main metabolite in faecal samples, the supernatant was
fractionated by chromatography on an L-column ODS with methanol-0.1% formic acid
(65:35, v/v). The isolated peak was identified by comparison of its HPLC retention time and
mass fragmentation pattern with the one of an authentic standard. The identified metabolite
was further incubated with recombinant human CYP2C8 protein as described previously. The
resulting metabolite peak, whose retention time was identical to that of the second most
abundant in faecal samples, was isolated by HPLC and identified by comparison of its HPLC
retention time and mass fragmentation pattern with the one of an authentic standard.
Chromatographic separation of metabolites was achieved using reversed-phase LC (an L-
column ODS, 5 μm, 150 × 4.6 mm ID, for LC/radiochromatography analysis and an L-
column ODS, 3 μm, 100 × 2.1 mm ID, for LC-MS/MS analysis). Phase A was
acetonitrile/10 mM ammonium acetate, pH 4.95 (10:90, v/v), phase B acetonitrile/10 mM
Accepted Manuscript
ammonium acetate, pH 4.95 (90:10, v/v). For LC/radiochromatography analysis, the
metabolites were eluted with the following linear gradient: equilibration at 20% B; 0 to 30
min, 20 to 70% B; 30 to 30.01 min, 70 to 100% B; 30.01 to 33 min, 100% B; and 33 to 33.01
min, 100 to 20% B. The flow rate was 1.0 mL/min in LC/radiochromatography analysis.
Radioactive metabolites were detected by mixing the eluent with Ultima Flo AP liquid
scintillant (3 mL/min) and passing it through a detection cell (500 μL) in the radiodetector.
The acquisition time was 30 min. Radiochromatograms generated from the radiometry were
processed using the FLO-ONE software package (version 3.6).For LC-MS/MS analysis , an
Acquity UPLC system with an L-column ODS (2.1 × 100 mm, 3 μm, Chemicals Evaluation
and Research Institute, Tokyo, Japan) coupled with an Acquity PDA UV detector and a
Waters Quattro Micro API mass spectrometer. The mobile phase was acetonitrile/10 mM
ammonium acetate, pH 4.95 (10:90, v/v) as solvent A and acetonitrile/10 mM ammonium
acetate, pH 4.95 (90:10, v/v) as solvent B. The flow rate was 0.3 mL/min and the column
temperature was 40°C. The drug and metabolites were eluted with the following gradient
program: starting from 20% B to 100% B at 10 min and maintained until 12 min; then back to
20% B at 12.01 min. The optimal tune parameters for the detection of selexipag and its
metabolites were as follows: capillary 3.5 kV; cone 30 V; extractor 2 V; source 120 °C; and
desolvation 400 °C. The scan range was set from 400 to 500. The MS data were processed
with MassLynx software (version 4.1, Waters).
Identification and quantification of metabolites circulating in human plasma
Human plasma samples from the 1800 µg b.i.d. and the 1600 µg b.i.d. dose group of a
clinical phase I multiple ascending dose study (Bruderer et al., 2014) were used to determine
the chemical structure and the amount of circulating metabolites, respectively. Once the
structural elucidation was performed, the metabolite quantification was subsequently done
Accepted Manuscript
with available synthetic references. Plasma, acidified with 1M HCl upon original preparation,
was pooled per time-point until 12 h post-dose and precipitated with acetonitrile containing
the internal standards ACT-293987B and ACT-333679B. Metabolites were investigated
using liquid chromatography combined with high resolution mass spectrometry and
radiodetection (split ratio MS/radiodetector, 1/5). The LC-MS system for metabolite
identification consisted of a Rheos Allegro (Flux Instruments) and a LC-10AD VP or LC-
20ADXR VP (Shimadzu) pump, an HTC PAL (CTC Analytics) autosampler and a Hot Dog
200/300 column oven (Thermo Fisher). Chromatographic separation was achieved using two
Gemini RP 150 x 2 mm columns with 3 µm particle size, in serial connection at 45°C. Eluent
A consisted of water containing 10 mM ammonium formate at pH 4.2, adjusted with formic
acid and eluent B consisted of acetonitrile with 0.1% formic acid. The flow rate was 0.2
mL/min. The following gradient was applied: starting from 20% B to 35% B at 5 min, then to
40% B at 15 min, to 60% B at 40 min, to 75 B at 47 min, to 95% B at 57 min maintained
until 58 min; then back to 20% B at 59 min. The HPLC was coupled with an LTQ Orbitrap
Discovery mass spectrometer equipped with a heated electrospray source H-ESI (Thermo
Fisher, San Jose, CA, USA) and operated under the following conditions: ESI potential 3 kV,
capillary temperature 270°C, vaporizer temperature 200°C, capillary 20.5 V, tube lens 100 V.
Sheath and auxiliary gas (nitrogen for both) were supplied using 60 and 5 arbitrary units
respectively. Full scan high resolution LC-MS chromatograms were recorded in positive ion
mode at a resolving power of 30000. Full scan high resolution LC-MS chromatograms were
generated to identify metabolites using the Metworks 1.2 and Xcalibur 2.0.7 software. The
determined exact mass error of all metabolites was below 3 ppm. Putative metabolites were
further investigated by MS/MS experiments in positive ion mode using collision induced
dissociation in the ion trap (CID) with 40% relative collision energy and high energy
collision dissociation (HCD) in the collision cell at 40 eV normalized collision energy as
Accepted Manuscript
fragmentation tools. On-line H/D exchange experiments were performed to investigate the
number of acidic protons for all metabolites using D
2
O instead of the water in eluent A. The
quantification of metabolites was carried out using an API5000 (AB Sciex Instruments,
Toronto, Canada) triple-stage quadrupole mass spectrometer equipped with a Turbo ionspray
interface operating in positive ion mode using qualified methods, calibration curves and
quality control samples.
Metabolism of selexipag and ACT-333679 in vitro
Hydrolysis of selexipag to ACT-333679
[
14
C]-selexipag was incubated at a final concentration of 10 µM in a 100 mM phosphate
buffer (pH=7.4, 37 °C) with pooled human liver microsomes (Xenotech, Kansas City, USA)
at a protein concentration of 1 mg/mL proteins. The reaction solutions without [
14
C]-
selexipag were pre-incubated at 37ºC for 5 min. The reaction was initiated by addition of
[
14
C]-selexipag and incubation was continued for 60 min at 37°C. Preliminary experiments
showed that the formation of [
14
C]-ACT-333679 from [
14
C]-selexipag was linear up to 60
min and 1 mg/mL of protein. The reactions were terminated by addition of one volume
equivalent of acetonitrile, and the samples were centrifuged at 16060 g for 5 min. An aliquot
of the supernatant was submitted to thin layer chromatography (TLC) analysis. Control
experiments in which acetonitrile was added to the reaction prior to [
14
C]-selexipag were run
under otherwise identical conditions to verify that ACT-333679 formation was indeed
enzyme-dependent.
In the inhibition experiments, carboxylesterase (CES) inhibitors bis(p-nitrophenyl) phosphate
(BNPP) and phenylmethylsulfonyl fluoride (PMSF), paraoxonase inhibitor
ethylenediaminetetraacetic acid (EDTA), and acetylcholine esterase inhibitor eserine were
added to the reaction mixture prior to initiating the reaction, each at concentrations of 0.01,
Accepted Manuscript
0.1 and 1 mM. [
14
C]-ACT-333679 produced in the reaction was determined by TLC. Silica
gel 60 F254, 0.25 mm (Merck, Darmstadt, Germany) was used as a TLC plate and a mixture
of dichloromethane, methanol and formic acid (10/2/0.1, v/v/v) as the developing solvent.
After development, the TLC plate was covered with a protective film (Saran Wrap, Asahi
Kasei Home Products, Tokyo, Japan). The TLC plate was in contact with an imaging plate
(TYPE BAS-MS, Fujifilm) and exposed in the lead shield box (BAS-SHB 2040, Fujifilm).
After exposure, image analysis was performed using a bio-imaging analyzer (BAS-2500) to
fractionate [
14
C]-selexipag, [
14
C]-ACT-333679 and other parts. The [
14
C]-ACT-333679
fraction (%)of the total radioactivity was used to calculate the formation rates of ACT-
333679 in the presence and in the absence of inhibitors. Finally, the remaining activity (%) in
the presence of inhibitors was calculated based on those rates.
Selexipag incubations with human liver preparations and plasma
[
14
C]-selexipag was incubated at 37 °C with pooled human liver microsomes (Biopredic
International, Rennes, France) containing 3 mg/mL proteins, in a 100 mM phosphate buffer
(pH=7.4) at a final concentration of 10 µM. The reaction was initiated by addition of the pre-
warmed NADPH-regenerating system and incubation was continued for 60 min at 37°C. In
the inhibition experiments with selexipag and sulfaphenazole (3 µM, CYP2C9), montelukast
(3 µM, CYP2C8), N-benzylnirvanol (5 µM, CYP2C19), quinidine (1 µM, CYP2D6), and
ketoconazole (1 µM, CYP3A4), an aliquot of concentrated inhibitor stock solution was added
to the reaction mixture prior to the initiation of the reaction. In the inhibition experiments
with the mechanism-based inhibitors azamulin (5 µM, CYP3A4), furafylline (20 µM,
CYP1A2) and ticlopidine (30 µM, CYP2B6), microsomes were first incubated for a period of
10 min at 37°C in the presence of the NADPH-regenerating system. The NADPH-
regenerating system was prepared as a 10-fold concentrated stock solution and kept at 20
Accepted Manuscript
°C. It consisted of 11 mM NADP, 100 mM glucose-6-phosphate and 50 mM magnesium
chloride in 100 mM phosphate buffer (pH 7.4). 20 UI/mL of glucose-6-phosphate
dehydrogenase were added in this mixture immediately before use. [
14
C]-selexipag was then
added at the end of the pre-incubation period and the reaction continued for another 60 min.
All experiments were performed in parallel in the presence or absence of 200 µM of the
carboxylesterase inhibitor BNPP (n=1). The organic solvent in all incubations was kept
below 1 % (v/v). The reactions were terminated by addition ice-cold acetonitrile, and the
samples were centrifuged at 20800 g for 10 min and 10 °C. An aliquot of the supernatant was
submitted to HPLC analysis. Control incubations in the absence of the NADPH regenerating
system or liver microsomes were performed in parallel under otherwise identical conditions.
Fresh human hepatocytes were provided in ready-plated cultures by Cytonet (Hannover,
Germany) and used immediately after receipt. They were cultured in fresh William's E
medium supplemented with 10 % foetal calf serum (v/v), 0.7 μM insulin, 10000 UI/mL
penicillin and 10 mg/mL streptomycin for 3 h prior to use according to the protocols provided
by the suppliers. At the end of the pre-incubation period, the medium was removed from each
well and replaced by pre-warmed (37 °C) William's E medium supplemented as described
above and additionally fortified with 1.0 μM hydrocortisone, 400 μM glutamine, and [
14
C]-
selexipag or [
14
C]-ACT-333679 at final concentrations of 10 μM. Triplicate wells were
sampled after up to 24 h of incubation by addition of methanol containing 0.1 M hydrochloric
acid and the entire well content was transferred into cryo-vials. Samples were stored frozen at
-20°C pending analysis.
Human plasma containing EDTA and prepared at Actelion Pharmaceuticals Ltd (Allschwil,
Switzerland) was fortified with [
14
C]-selexipag at a final concentration of 10 μM. After an
incubation time of up to 6 h, plasma proteins were precipitated by addition of an ice-cold
Accepted Manuscript
mixture of acetonitrile and methanol. The samples were centrifuged at 20800 g for 10 min at
10 °C, and a 450 μL-aliquot of the supernatant was submitted to HPLC analysis.
Selexipag incubations with recombinant cytochrome P450 enzymes
Incubations of [
14
C]-selexipag with recombinant human P450 enzymes CYP1A2, 2B6, 2C8,
2C9, 2C19, 2D6 and CYP3A4 were performed at a single concentration of 10 µM. An aliquot
of the [
14
C]-selexipag stock solution was added to a 100 mM phosphate buffer (pH 7.4, 37
°C) containing the P450 enzyme at a concentration of 100 pmol/mL. A co-incubation of
recombinant CYP1A2 and CYP3A4 was also performed and contained each enzyme at a
concentration of 100 pmol/mL under otherwise same experimental conditions. The reaction
was initiated by adding pre-warmed NADPH-regenerating system and incubation was
continued for 60 min at 37°C in an Eppendorf thermomixer at 650 rpm. The reaction was
terminated by addition of one volume equivalent of ice-cold acetonitrile. The samples were
centrifuged at 20800 g for 10 min and 10°C. A 100 µL aliquot of the supernatant was
submitted to HPLC analysis. Control incubations in the absence of the NADPH-regenerating
system, as well as incubations with control bactosomes without P450 enzymes were
performed in parallel under otherwise identical conditions (n = 1).
Analytical method for selexipag incubations
Metabolic profiles from incubations with human liver fractions and recombinant enzymes
were analysed using HPLC coupled with radioactivity detection. The analytical system
consisted of two Shimadzu HPLC pumps LC-30AD (Shimadzu, Reinach, Switzerland)
equipped with a Shimadzu membrane degasser DGU-20A5, a Shimadzu diode array detector
SPD-M20A, a Shimadzu column oven CTO-20, a Berthold radioflow detector LB513 with a
200 µL-liquid cell Z-200-6M, a LB5036 pump for supplementing liquid scintillation cocktail
at 3 mL/min (Berthold AG, Regensdorf, Switzerland), and a Shimadzu autosampler model
Accepted Manuscript
SIL-30AC. Radiochemical data acquisition was done using the RadioStar software (version
5.0.12.4, Berthold AG, Regensdorf, Switzerland). For the chromatographic analysis of [
14
C]-
selexipag and its metabolites, two different HPLC gradients were used. The first 67-min
gradient used 20, 35, 40, 60 75, 95, 95, 20 and 20% of mobile phase B at 0, 5, 15, 40, 47, 57,
58, 59 and 67 min run time, respectively. The second 90-min gradient used 20, 35, 40, 60, 62,
95, 95, 20 and 20% phase B at 0, 5, 15, 40, 80, 81, 82, 83 and 90 min run time, respectively.
The chromatographic separation was achieved using a Phenomenex Gemini C18 column
(5 µm, 250 x 4.6 mm ID, 110 Å) combined with a Phenomenex security guard cartridge
Gemini C18 (4 x 3 mm ID) at 45 °C with a flow rate of 1.0 mL/min. Mobile phases consisted
of 10 mM ammonium formate adjusted to pH 4.1 with formic acid (phase A) and acetonitrile
containing 0.1 % formic acid (phase B). Serial UV detection in a wavelength range of 190-
500 nm and
14
C -radiochemical detection were performed.
[
14
C]-ACT-333679 incubations with human liver microsomes, recombinant cytochrome
P450 and UGT enzymes
[
14
C]-ACT-333679 was incubated with pooled liver microsomes BD Biosciences (Tokyo,
Japan) containing 1 mg/mL proteins, in a 100 mM phosphate buffer (pH=7.4, 37 °C) at a
final concentration of 10 and 100 µM. The reaction was initiated by addition of the pre-
warmed NADPH-regenerating system and incubation was continued for 60 min at 37°C. In
the inhibition experiments with [
14
C]-ACT-333679 and furafylline (20 µM, CYP1A2), 8-
methoxypsoralen (5 µM, CYP2A6), sertraline (10 µM, CYP2B6), quercetin (5 µM,
CYP2C8), sulfaphenazole (3 µM, CYP2C9), N-benzylnirvanol (5 µM, CYP2C19), quinidine
(1 µM, CYP2D6), diethyldithiocarbamate (100 µM, CYP2E1) and ketoconazole (1 µM,
CYP3A4/5), each specific inhibitor methanol solution was placed into a polypropylene tube
and evaporated to dryness under a stream of nitrogen gas. Diethyldithiocarbamate aqueous
solution was used without dryness. The buffer and microsomes were added to the specific
Accepted Manuscript
inhibitor and stirred using a vortex mixer. The mixture was mixed with the test substance
solution and pre-incubated at 37°C for 5 min. NADPH regenerating system cofactor solution
was added to the mixture to start the reaction and incubated at 37°C for 1 h. As negative
controls, a NADPH regenerating system non-added group and specific inhibitor non-added
group were treated in the same way. Incubations of [
14
C]-ACT-333679 with recombinant
human P450-expressing microsomes CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and
CYP3A4 (50 pmol/ml) were performed at a single concentration of 10 µM and 100 µM in
100 mM phosphate buffer (pH 7.4, 37 °C) in duplicate. Similarly, [
14
C]-ACT-333679 was
incubated with recombinant UGT enzymes: UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9,
1A10, 2B4, 2B7, 2B15 and UGT2B17 (1 mg proteins/mL) in 100 mM phosphate buffer (pH
6.0, 37 °C) in duplicate. The P450 reactions were initiated by adding the NADPH-
regenerating system in the pre-incubated mixture and incubation was continued for 1 h at
37°C. Control incubations in the absence of the NADPH-regenerating system, as well as
incubations with control microsomes without P450 enzymes at 1 mg/mL protein
concentration were performed in parallel under otherwise identical conditions. All reactions
were terminated by addition of two volumes equivalent of ice-cold methanol. The samples
were centrifuged at 22000 g for 5 min at 4°C and the procedure was repeated once with the
pellet. Supernatants were combined, evaporated to dryness and reconstituted in 350 μL of the
HPLC initial mobile phase. A 50 µL-aliquot of the supernatant was submitted to HPLC
analysis. The UGT reactions were initiated by adding a UDPGA cofactor solution (2 mM
UDPGA, 50 g /mL alamethicin, 5 mM D-saccharic acid 1,4-lactone monohydrate, and 10
mM MgCl
2
as final concentrations) in the pre-incubated mixture and incubation was
continued for 30 min at 37°C. The reaction was terminated by addition of two volumes
equivalent of ice-cold methanol containing hydrochloric acid. The samples were centrifuged
at 22000 g for 5 min at 4°C and the procedure was repeated once with the pellet.
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Supernatants were combined, evaporated to dryness and reconstituted in 350 μL of the HPLC
initial mobile phase. A 50 µL-aliquot of the supernatant was submitted to HPLC analysis.
Control incubations in the absence of the UDPGA cofactor solution, as well as incubations
with control microsomes without UGT enzymes at the highest protein concentration were
performed in duplicate in parallel under otherwise identical conditions.
Analytical method for ACT-333679 incubations
Metabolic profiles were analysed using HPLC coupled with radioactivity detection. The
analytical system consisted of a delivery unit LC-10AD (Shimadzu, Kyoto, Japan), a
Shimadzu system controller SCL-10A, a Shimadzu UV-VIS detector SPD-10A, a Shimadzu
column oven CTO-10AS, a Shimadzu radioactivity detector (RAD) 525TR and a Shimadzu
autosampler model SIL-HTC. Radiochemical data acquisition was done using ChemStation
(Agilent) and the Flo-One software package version 3.65 built in the RAD. The
chromatographic separation of [
14
C]-ACT-333679 and its metabolites was achieved using a
Chemicals Evaluation and Research Institute L-column ODS (5 μm, 4.6 × 150 mm) at 40°C
with a flow rate of 1.0 mL/min. Mobile phases consisted of 0.1% formic acid in water (phase
A) 0.1% formic acid in methanol ( phase B). The applied gradient method started at 0 min
with 60% mobile phase B increased to 90% at 15 min and maintained until 25 min, then back
to 60% at 25.01 min and maintained until 30 min. UV detection at a wavelength of 254 nm
and radiochemical detection were performed.
Inhibition of cytochrome P450 enzymes
The potential of selexipag and its metabolite ACT-333679 to inhibit cytochrome P450
enzymes was studied in vitro using human liver microsomes (Xenotech LLC, Nosan
corporation, Japan) containing 0.2 mg/mL proteins in a 100 mM phosphate buffer (pH=7.4,
37 °C).The reaction was initiated by addition of the NADPH-regenerating system containing
Accepted Manuscript
the glucose-6-phosphate dehydrogenase and continued for 2-30 min at 37 °C. The following
P450 isoform-specific marker transformations and the substrate concentrations were used:
phenacetin-O-deethylation (CYP1A2, 6 µM), coumarin 7-hydroxylation (CYP2A6, 0.2 µM),
bupropion hydroxylation (CYP2B6, 20 µM), paclitaxel 6-hydroxylation (CYP2C8, 2 µM),
diclofenac 4'-hydroxylation (CYP2C9, 1 µM), (S)-mephenytoin 4'-hydroxylation (CYP2C19,
4 µM), bufarolol 1'-hydroxylation (CYP2D6, 2 µM), chlorzoxazone 6-hydroxylation
(CYP2E1, 8 µM), and midazolam 4'-hydroxylation (1 µM) and testosterone 6-hydroxylation
(10 µM) for CYP3A4. The biotransformation was assessed by product formation. Metabolite
formation was assessed by LC/MS-MS using qualified methods, calibration curves and
quality control samples. The following positive controls were used: -naphtoflavone
(CYP1A2, 0.2 µM), 8-methoxypsoralen, (CYP2A6, 0.2 µM), ticlopidine (CYP2B6, 0.2 µM),
quercetin (CYP2C8, 25 µM), sulfaphenazole (CYP2C9, 5 µM), tranylcypromine (CYP2C19,
25 µM), quinidine (CYP2D6, 1 µM), diethyldithiocarbamate (CYP2E1, 20 µM),
ketoconazole (CYP3A4, 0.5 µM). Selexipag and ACT-333679 were tested up to 50 µM in
duplicate. Samples were analysed using LC-MS/MS except for CYP2A6 (HPLC). The IC
50
values were calculated by Pharmacodynamic model 103 (Inhibitory effect E
max
model) with a
pharmacokinetic analysis software product, WinNonlin Version 5.2 (Pharsight Corporation).
The K
i
values were calculated by plotting (Dixon plot) the reciprocal metabolite formation
rates on the y-axis and test substance concentrations on the x-axis, followed by the analysis
with appropriate model formula using WinNonlin Ver. 5.2.1 (Pharsight). Time-dependent
inhibition was assessed on CYP2C8, CYP2C9, CYP2D6, and CYP3A4 using human liver
microsomes and comparing IC
50
values obtained with and without a pre-incubation period.
The so-called IC
50
shift is a marker of the extent of change in enzyme activity during the pre-
incubation period.
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Induction of cytochrome P450 enzymes
Changes in CYP1A2, CYP2C9, CYP2B6, and CYP3A4 mRNA expression were assessed in
at least three different batches of freshly isolated or cryopreserved human hepatocytes,
obtained from Biopredic International (Rennes, France) and Bioreclamation IVT (Neuss,
Germany), respectively. Hepatocytes were incubated in triplicate wells with either DMSO
(control) or selexipag or ACT-333679 at concentrations ranging from 0.1 to 100 µM. The
positive controls were rifampicin for CYP3A4 and CYP2C9 (0.1-50 µM), phenobarbital for
CYP2B6 (100-3000 µM) and omeprazole for CYP1A2 (1-100 µM). Compound solutions were
prepared by diluting the respective DMSO stock solutions with cell culture medium by a factor of
1000, i.e. with 0.1% of DMSO in the incubation medium.. All incubations were done for 68 h
using cell culture media with 0.2% bovine serum albumin. The hepatocytes lysates were used
for total RNA extraction with a Qiagen Rneasy micro kit (Qiagen AG, Switzerland). DNase
(recombinant human desoxyribonuclease purchased from Qiagen) treatment was applied
during the RNA extraction process, to avoid contamination by genomic DNA.
Complementary DNA (cDNA) was synthesized using 50 ng of total RNA as template with
the Taqman reverse transcription reagents kit from Life Technologies Europe B.V. (Zug,
Switzerland) and MultiscribeTM reverse transcriptase at 1.25 IU/μL, random hexamers at 2.5
μM, and RNase inhibitor at 0.4 IU/μL. PCR was performed using the Taqman PCR
Mastermix from Life Technologies. CYP3A4, CYP2C9, CYP1A2, CYP2B6 and 18S
transcript levels were quantified with a StepOnePlus sequence detection system (Life
Technologies) according to the manufacturer’s protocol. Relative transcript levels in
compound-treated cells and DMSO-treated controls were determined using the comparative
Ct (cycle threshold) method (ΔΔCt method). The amount of target, normalized to an
endogenous control (18S) and relative to a calibrator (control sample in the experiment, i.e.
DMSO-treated hepatocytes) is given by 2-ΔΔCt. The fold-induction for each gene and
compound concentration was calculated using the relative transcript level ratio over the one
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obtained for DMSO-treated cells. Statistical analysis and curve fitting for EC
50
determinations were performed using the GraphPad Prism software (version 5, GraphPad
Software Inc., San Diego, USA). For the construction of the concentration-response curves, a
three-parameter equation (sigmoid) was used Y = Bottom + (Top-Bottom)/(1 + 10((log EC50
- X), in which X equals the logarithm of concentration, and Y equals the fold-induction.
Inhibition of drug transporters
The effect of selexipag and ACT-333679 on the activity of the multidrug resistance protein
MDR1 (ABCB1, P-gp) was investigated using MDR1-overexpressing MDCK-II cells
(Netherland Cancer Institute, Amsterdam, Netherlands). Tritiated digoxin (1 M) and
rhodamine 123 (10 M) were used as substrates. Inhibition of P-gp by selexipag and ACT-
333679 were tested at concentrations up to 10 M. Experiments with digoxin were analysed
using a liquid scintillation analyser with luminescence correction and on-line quenching
correction by means of an internal standard (LSC) and those with rhodamine 123 by
spectrofluorimetry. Verapamil was used as a control inhibitor (100 µM). Apparent
permeability coefficients (P
app
) and net secretory flux were calculated as previously described
(Fenner et al., 2009). Inhibition of the efflux transporters BCRP, BSEP and MRP2 was
investigated in transporter-overexpressing Sf9 vesicles obtained from SOLVO Biotechnology
(Budaörs, Hungary). The tritiated model substrates methotrexate (100 M), taurocholic acid
(5 M) and estradiol-17-β-glucuronide (50 M) were used for BCRP, BSEP and MRP2
together for selexipag and ACT-333679 up to 1000 M. Control inhibitors were Ko143 (0.3-
2 µM), cyclosporine A (0.5-5 µM) and MK571 (1-100 µM), respectively. Samples were
analysed by LSC. Inhibition of the uptake transporters OATP1B1, OATP1B3 and OAT1 by
selexipag and ACT-333679 was investigated in Chinese hamster ovary (CHO) cells
overexpressing the respective transporters. The OATP1B1/1B3 overexpressing cells were
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kindly provided by Professor B. Stieger (University of Zurich, Switzerland) and the OAT-1
cells were from SOLVO Biotechnology (Budaörs, Hungary). The tritiated model substrates
atorvastatin (0.5 M), taurocholic acid (5 M) and p-aminohippuric acid (1 M) were used
together with selexipag and ACT-333679 up to 100 µM and 1000 M. The positive controls
used were cyclosporin A (OATP1B1/1B3, 5 µM) and probenecid (OAT1, 5-100 µM).
Samples were analysed by LSC. Inhibition of the uptake transporters OAT3, OCT1 and
OCT2 and the efflux transporters MATE1 and MATE2K was investigated in transporter-
overexpressing human embryonic kidney (HEK293) cells, all from Absorption Systems LP
(Exton, USA). The respective model substrates and control inhibitors were: furosemide (5
M) and probenecid (100 µM); 1-methyl-4-phenylpyridinium iodide (MPP, 5 µM, for OCT1
and OCT2) and repaglinide (600 µM) and imipramine (300 µM) ; metformin (50 M) and
cimetidine (100 µM); 4-(4-(dimethylamino)-styryl)-N-methyl-pyridinium (ASP, 1 M).
Selexipag and ACT-333679 were tested at concentrations up to 100 M. Samples were
analysed using LC-MS/MS. For each transporter studied, the respective substrate incubation
period was selected so that transport was linear over time. All incubations were performed
either in duplicate or in triplicate in one or two independent experiments. Cellular and
vesicular uptake rates were normalized to total protein content and incubation. Cellular net
uptake rates were calculated as the difference in the uptake rate of the overexpressing cells
and corresponding wild-type cells. For the studies with BCRP, MRP2 and BSEP
overexpressing vesicles, transporter-mediated net uptake rates were calculated as the
difference of the values obtained in the absence and presence of adenosine triphosphate.
All data were evaluated by plotting the inhibitor concentration (logarithmic scale) against the
net uptake rate. IC50 values were determined from the plot by non-linear regression using he
GraphPad Prism software package (version 5.0, GraphPad Software Inc., La Jolla, CA) and
the following equation: Y = ((Top/(1+ (x/IC
50
)
s
) + Bottom, where y is the net uptake rate, x is
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the inhibitor concentration (µM), s is the slope at the point of inversion, and Top and Bottom
the maximum and minimum values for the cellular uptake rate or % of control.
In vitro binding of selexipag and ACT-333679
The binding of selexipag and/or ACT-333679 was determined in human plasma, human liver
microsomes, and in the incubation media used in the in vitro perpetrator investigations on
cytochromes P450 and drug transporters. Human plasma containing EDTA and prepared at
Covance Laboratories Ltd (Harrogate, England) was fortified with [
14
C]-selexipag or [
14
C]-
ACT-333679 in a concentration range of 0.1-50 g/mL (0.2-100 nM or 0.24-120 nM,
respectively) . Equilibrium dialysis was performed using a Dianorm equilibrium dialyser
(Pierce and Warriner, Chester, UK) at 37°C against 10 mM phosphate-buffered saline (pH
7.4) for 4 h, the time at which equilibrium was reached. Following dialysis, aliquots were
taken from the donor and buffer compartment, and total radioactivity was quantified by LSC.
The binding in human liver microsomes and incubations media were determined by
equilibrium analysis using a Pierce Rapid Equilibrium Dialysis (RED) device (Thermo Fisher
Scientific, Lausanne, Switzerland) and non-labelled selexipag or ACT-333679. Incubations
were carried out on a shaker at 37°C under 5% CO
2
atmosphere for 4 h. The donor
compartment was either 1 mg/mL human liver microsomes, vesicles or cell culture medium.
Donor and receiver (containing phosphate buffer, pH 7.4) were analysed by LC-MS/MS and
the free fraction and recovery were calculated.
Estimation of [I, gut] for selexipag
[I, gut], i.e. calculated gut concentration, was estimated following the current European
medicines agency (EMA) and the food and drug administration (FDA) guidelines for drug-
drug interactions (EMA, 2012; FDA, 2012) for cytochrome P450 enzymes, and as described
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previously for P-gp/MDR1 (Agarwal S, Arya V and Zhang L, 2012). The equation used was
[I, gut] = f
a
k
a
Dose/Q
en
, where f
a
is the fraction absorbed, k
a
the absorption rate constant,
and Q
en
the blood flow through the enterocytes. The absorption rate constant was assumed to
be 1 and for the blood flow through the enterocytes, a value of 300 mL/min was used. The
fraction absorbed was determined using Phoenix WinNonlin v.6 (Certara, Princeton, NJ,
USA) based on observed plasma concentration time profiles of selexipag after oral dosing
using 1-compartamental modelling (Kaufmann et al., 2017).
Results
The metabolism and excretion of selexipag has been investigated in human after oral dosing
of
14
C-labeled drug and sampling of urine and faeces over time intervals up to 168 h to
determine the total radioactivity eliminated and allow the analysis of metabolites (Kaufmann
et al., 2012). Total radioactivity was eliminated primarily in the faeces, accounting for 92.7%
of the administered dose with 87.6% of the administered dose recovered within 96 h. Renal
excretion of drug-related material accounted for 11.9% of the dose. Overall, total
radioactivity recovered in urine and faeces represented 104.6% of the administered dose.
To determine the nature of the metabolites eliminated via the faeces and due to the low
absolute radioactivity contained in individual samples, those extracts with the highest
radioactivity were selected for further analysis. Metabolic patterns were recorded using
HPLC coupled to radiodetection. A representative chromatogram of a faecal sample collected
during a 24-48 h interval from a healthy subject is depicted in Figure 2A. Besides
ACT-333679, the main product P10 was observed, followed by P18. Selexipag was absent
from all faecal samples analysed. The identity of metabolites for which synthetic standards
were available (P10 and P18) was confirmed by comparing their chromatographic retention
times and MS/MS spectra.. [
14
C]-ACT-333679 and ACT-333679 were first incubated with
recombinant human CYP2C8 and each putative radiolabeled and unlabeled metabolite P10
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isolatedChromatographic retention times of these isolated metabolites were identical to the
one of metabolite P10 in human faecal samples. The key fragment ions generated were m/z
394, 378, 318, 276 and 249 and were in agreement with the fragmentation pathways of the
respective authentic standard (Figure 2B-C). P10 was identified as a product of
hydroxylation at the phenyl ring of ACT-333679. Similarly, isolated radiolabeled and
authentic standard P10 were further incubated with recombinant CYP2C8 and each putative
radiolabeled and unlabeled metabolite P18 isolated. The resulting product, which retention
time was identical to that of P18, was isolated by HPLC and unequivocally identified by
comparison of its HPLC retention time and mass fragmentation pattern with those of an
authentic standard (Figure 2, D-E). P18 was identified as a hydroxylation product of P10. In
conclusion, P10 was identified as a product of hydroxylation at the phenyl ring of ACT-
333679 and was further hydroxylated at the isopropyl group to form P18.
Due to the low total radioactivity content in the urine and plasma samples from the
radioactive study in man, no metabolite identification studies could be performed on them.
Therefore, time-pooled plasma samples from a separate clinical phase I study following
multiple doses of 1600 µg and 1800 µg b.i.d (Bruderer et al., 2014) were used to determine
the nature and the amount of circulating metabolites by LC-MS/MS analysis. For the
metabolite identification work, human plasma samples from the 1800 µg dose group were
initially pooled per time point. In addition to the already known metabolite ACT-333679,
metabolites P4, P10, P11, P12, P13, P14, P34 and P35 were identified. The proposed
structures of these metabolites were confirmed by comparison with chemical references
which displayed identical retention time and MS fragmentation pattern. The quantification of
these metabolites was carried out using LC-MS/MS and an additional set of human plasma
samples from the 1600 µg dose group pooled per time point at: pre-dose, 2 h, 4 h, 6 h and 12
h post-dose using known standards. The relative abundance of selexipag and its metabolites
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in human plasma, as well as the determined and exact theoretical mass values for the
metabolites are summarized in Table 1. Based on the AUC
0-last
values, selexipag and
ACT-333679 were the most abundant entities representing 14% and 78% of drug-related
material, while none of the other metabolites (P4, P10-P14, P34 and P35) exceeded 3% of
drug-related material.
The identification of enzymes involved in the biotransformation of selexipag was performed
in vitro using various biological preparations of human origin, including plasma, hepatocytes,
liver microsomes and also recombinant phase I and phase II enzymes.
To determine the nature of the hydrolytic enzymes involved in the formation of ACT-333679
from selexipag, human liver microsomes were used in the presence and in the absence of
hydrolase inhibitors. BNPP and PMSF were used as carboxylesterase (CES) inhibitors,
EDTA as a paraoxonase inhibitor and eserine as an acetylcholine esterase inhibitor.
Hydrolysis of selexipag leading to the formation of ACT-333679 was absent from
incubations in human plasma. The effect of the various inhibitors on the formation rate of
ACT-333679 from selexipag in human liver microsomes is shown in Figure 3. Incubations in
the absence of the NADPH-regenerating system yielded significant amounts of ACT-333679,
i.e. with a formation rate between 13-19 pmol/min/mg microsomal protein. Hydrolysis, i.e.
ACT-333679 formation, was suppressed in the presence of the CES inhibitors BNPP and
PMSF, whereas the paraoxonase and acetylcholine esterase inhibitors EDTA and eserine had
little or no effect.
The human P450 enzymes catalyzing the metabolism of selexipag and ACT-333679 were
investigated as follows: two complementary approaches were pursued, i.e., incubation with
liver microsomes in the absence or presence of chemical inhibitors for the various P450
isoforms, and incubation with recombinant P450 enzymes. Parallel sets of experiments were
performed with and without the esterase inhibitor BNPP to suppress hydrolysis of selexipag
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and its metabolites by microsomal enzymes. Incubations with human hepatocytes were also
performed. Metabolic profiles were recorded using HPLC coupled to radiodetection.
Representative radiochromatograms of incubations with human liver microsomes,
recombinant CYP2C8, CYP3A4, UGT1A3 or UGT2B7 are illustrated in Figures 4-6.
Metabolites generated after incubations in human liver microsomes or hepatocytes are
summarized in Table 2.
Formation of metabolites P8, P10, P14, P36, P39, P40 and ACT-333679 was observed after
the incubation of [
14
C]-selexipag with liver microsomes representing altogether about 30% of
the respective radiochromatograms. ACT-333679 was the most prominent metabolite. Only
ACT-333679 and P10 were affected by the presence of BNPP suggesting a role of
carboxylesterase in their formation. The CYP2C8 inhibitor montelukast reduced P8
formation and completely inhibited P39 formation. Montelukast reduced by half the
formation of P10. The CYP3A4 inhibitors azamulin and ketoconazole led to a reduction in
P40 formation. Metabolite P36 formation was completely inhibited in the presence of
azamulin whereas its formation was only reduced by ketoconazole. Metabolite P14 was not
detected in experiments with azamulin. Inhibition of P14 formation by ketoconazole resulted
only in a partial reduction. The formation of P14 was not detected in presence of the
CYP1A2 selective inhibitor furafylline. Incubations in the combined presence of
montelukast, ketoconazole and BNPP were also investigated in order to assess the overall
contribution of CYP2C8, CYP3A4 and carboxylesterase in selexipag metabolism. The
formation of metabolites P10, P14, P36, P39 and P40 was completely inhibited by
combination of the three inhibitors. P8 formation was reduced by 60%. When [
14
C]-selexipag
was incubated with recombinant P450 enzymes, turnover was mostly observed with CYP2C8
and CYP3A4. The formation of P8 and P39 was only seen with CYP2C8. Formation of P14,
P36, P40 and P41 was observed in the presence of CYP3A4. The formation of P41 was
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decreased in the combined presence of CYP3A4 and CYP1A2. Conversely, P14 formation in
presence of CYP3A4 was increased in the presence of the CYP3A4/CYP1A2 combination.
When [
14
C]-ACT-333679 was incubated with human liver microsomes, P10 was by far the
main metabolite observed and its formation was reduced in the presence of the CYP2C8
inhibitor quercetin. The incubation of [
14
C]-ACT-333679 with recombinant CYP2C8 lead to
the abundant formation of P10, which was not observed with any other recombinant P450
tested. Also, using recombinant CYP3A4 and [
14
C]-ACT-333679, several products were
observed that were not structurally identified. Since P11, the acylglucuronide of ACT-
333679, was identified in small amounts in human plasma, [
14
C]-ACT-333679 was incubated
with a battery of recombinant UGT enzymes. The formation of P11 was mostly observed in
the presence of UGT1A3 followed by UGT2B7.
The binding of selexipag and ACT-333679 to human plasma proteins was investigated using
equilibrium dialysis. Mean plasma binding values of selexipag and ACT-333679 were 99.7 %
and 99.6 %, respectively.
The potential of selexipag and ACT-333679 to inhibit cytochrome P450 enzymes was studied
in vitro using human liver microsomes and P450 isoform-specific marker transformations.
Time-dependent inhibition was assessed for both compounds on CYP2C8, CYP2C9,
CYP2D6 and CYP3A4 using human liver microsomes. The results of the competitive
inhibition experiments and the derived free inhibition constant (K
i
) values are summarized in
Table 3. Selexipag only weakly affected most of the human P450 enzymes. IC
50
values were
around or above 15 M for CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP2E1 and
CYP3A4. There was stronger inhibition of two members of the CYP2C family. K
i
values for
CYP2C8 and CYP2C9 were 0.6 M and 1.1 M. ACT-333679 was a less potent inhibitor.
With the exception of CYP2C8 and CYP2C9, IC
50
values for all P450 enzymes were above
15 M. K
i
values for CYP2C8 and CYP2C9 were 7.7 M and 10.5 M. Neither selexipag
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nor ACT-333679 elicited a time-dependent inhibition of CYP2C8, CYP2C9, CYP2D6, or
CYP3A4.
The potential of selexipag and ACT-333679 to induce cytochrome P450 enzymes was studied
in vitro using human hepatocytes. Changes in CYP1A2, CYP2C9, CYP2B6, and CYP3A4
mRNA expression were assessed in different batches of primary human hepatocytes. The
results of these experiments and the derived free EC
50
values are summarized in Table 4.
Both compounds elicited concentration-dependent increases in CYP3A4, CYP2C9, and
CYP2B6 mRNA. Selexipag and ACT-333679 had no relevant effect on CYP1A2 mRNA.
The corresponding mean EC
50
values for selexipag on CYP3A4, CYP2C9 and CYP2B6
mRNA were 27 nM, 7 nM and 140 nM. ACT-333679 was generally less potent and the
values were 100 nM, 30 nM and 130 nM.
The potential of selexipag and ACT-333679 to inhibit drug transporters was assessed in cells
and membrane vesicles overexpressing the protein of interest. The results of these
experiments and the derived free IC
50
values are summarized in Table 5. Selexipag inhibited
the uptake transporters, OCT1 and OCT2, and the efflux transporters P-gp/MDR1, BSEP,
MATE1, MATE2K, and MRP2 with IC
50
values ranging from 8 µM to above 80 μM.
Stronger inhibition was observed on the uptake transporters OATP1B1, OATP1B3, OAT1,
and OAT3 with IC
50
values in the range of 1.0-1.7 µM. Selexipag showed a similar inhibition
of the efflux transporter BCRP with an IC
50
of 0.8 µM. ACT-333679 was generally less
potent. Except for OATP1B1, OATP1B3, BCRP, and OAT3, IC
50
values were higher than 10
µM. IC
50
values for OATP1B1, OATP1B3, BCRP, and OAT3 were in a range from 1.7-3.3
µM.
To assess the potential for selexipag to cause drug-drug interactions in the gastrointestinal
tract, [I,gut] was calculated. . [I, gut] after the maximal selexipag dose of 1600 µg was 81
ng/mL (Table 6).
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Discussion
The metabolism pathways of selexipag, investigated in human after oral dosing and
completed by in vitro studies in various enzymatic systems, are depicted in Figure 7.
Selexipag undergoes several primary biotransformation reactions: hydrolysis of the
sulfonamide leads to the formation of the pharmacologically active ACT-333679. Aromatic
hydroxylation of the pyrazine ring produces P36, while hydroxylation at the phenyl ring gives
rise to P8. Hydroxylation at the isopropyl group produces P39. Dealkylation of the
aminopyrazine resulting in the loss of the isopropyl moiety yields P40. An unusual ring
contraction of the pyrazine to an imidazole ring produces P35. Finally, another dealkylation
of the aminopyrazine resulting in the loss of the butoxy-N-(methanesulfonyl)acetamide
moiety yields P41. Most primary metabolites undergo subsequent hydrolysis to their
corresponding acids P4, P10, P12, P13 and P34. Further dealkylation of P41 leads to the
formation of P14. ACT-333679 is conjugated with glucuronic acid to the acyl-glucuronide
P11, and hydroxylated at the phenyl ring to form P10. P10 is then further oxidized at the
isopropyl group to yield P18.
The main metabolic pathway of selexipag is the formation of the active metabolite ACT-
333679. Except for ACT-333679, none of the circulating metabolites in human plasma
exceed 3% of the total drug-related material. The inhibition of ACT-333679 formation in
human liver preparations by the CES inhibitors as well as the absence of hydrolysis of
selexipag in human plasma (Li et al., 2005) indicate a role for hepatic CES enzymes. The
human P450 enzymes involved in selexipag metabolism have been identified. In addition to
ACT-333679, five primary metabolites of selexipag were formed. Metabolites P8 and P39
were formed by CYP2C8 whereas metabolites P36, P40 and P41 were formed by CYP3A4.
Metabolite P41 was N-dealkylated to the secondary metabolite P14 by CYP1A2. The
experiments on P450 enzymes involved in the metabolism of ACT-333679 focused on P10
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formation as the main metabolite in human faeces. CYP2C8 was the only P450 isoform
catalyzing the aromatic hydroxylation to P10. CYP2C8 together with CYP3A4 were also
involved in the formation of several minor ACT-333679 metabolites. There was evidence
that CES contributed to the formation of the secondary metabolite P10 from metabolite P8.
UGT1A3 and UGT2B7 catalyzed the glucuronidation of ACT-333679 to the acyl
glucuronide P11.
The human pharmacokinetics of selexipag after oral dosing have been described in several
studies (Bruderer et al., 2014; Baldoni et al., 2015; Hoch et al., 2015). Selexipag is rapidly
absorbed and is hydrolysed to its active metabolite ACT-333679. Maximum observed plasma
concentrations of selexipag and ACT-333679 are reached within 13 h and 34 h,
respectively. A study after intravenous dosing of 200 µg selexipag was performed to
determine its absolute bioavailability which was 49 % (Kaufmann et al., 2017). In healthy
subjects and in patients with PAH, plasma exposure to ACT-333679 at all doses and at
steady-state is approximately 3-to 4-fold higher than to the parent compound. In the study
after intravenous dosing of selexipag, the mean exposure ratio of ACT-333679 to selexipag
was approximately 1.3. These data indicate a pre-systemic formation of ACT-333679,
potentially by intestinal CES. Indeed, it is known that in man CES enzymes are expressed in
the liver and in the gut and exist as different isoenzymes. CES1 is mostly expressed in the
liver, while CES2 is present in the liver and the intestine (Yang et al., 2009). A pre-systemic
contribution of CES2 to the formation of ACT-333679 is therefore likely.
The potential of selexipag and ACT-333679 as perpetrators of metabolic- and/or transporter
driven drug interactions was studied in vitro. In order to connect the in vitro data to the
selexipag and ACT-333679 exposures observed in the clinic, two separate assessments were
done. The first one considered the potential perpetrator effects on P450 enzymes and
transporters in liver and kidney, the second one in the intestine. For the first assessment,
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knowledge of total peak plasma concentrations of selexipag and ACT-333679 were used. At
the highest treatment dose of 1600 g, these were 19.8 ng/mL and 28.7 ng/mL, i.e., about
40 nM and 70 nM for selexipag and ACT-333679. The respective free concentrations
calculated after correction for plasma protein binding were 0.12 nM and 0.28 nM. The
strongest inhibitory effects for selexipag/ACT-333679 on P450 enzymes were observed on
CYP2C8 and CYP2C9 Table 3). Similarly, the strongest inductive effects for
selexipag/ACT-333679 were seen on CYP3A4 and CYP2C9 (Table 4). These in vitro
concentrations are several magnitudes higher than the free observed plasma concentrations
rendering such interactions unlikely to happen in the clinic. In support of this assessment, no
change in the exposure to S-warfarin (CYP2C9 substrate) or R-warfarin (CYP3A4 substrate)
was observed in a healthy subjects study where selexipag was dosed at 400 g b.i.d. after a
single dose of 20 mg warfarin (Bruderer et al., 2016). Finally, for the inhibition of uptake and
efflux transporters, the strongest inhibitory effects for selexipag and ACT-333679 were
observed on OATP1B1, OATP1B1B3, OAT1, OAT3 and BCRP and were between 0.8-1.7
µM and 1.7-20 µM (Table 5). For the same reasons as indicated above for P450 enzymes,
such interactions are unlikely to happen.
For the second assessment dealing with the potential inhibitory effect of selexipag on
intestinal P450 enzymes and transporters, the gut concentration was calculated according to
the current EMA and FDA guidelines (EMA, 2012; FDA, 2012), i.e. using calculated gut
concentrations [I,gut] and comparing them to the IC
50
for BCRP inhibition, or EC
50
for
CYP3A4 induction. The [I,gut] after the maximal selexipag dose of 1600 µg was calculated
to be 81 ng/mL (Table 6), which is much lower than the IC
50
for BCRP (400 ng/mL) and also
less than 0.1 times higher than the EC
50
for CYP3A4 induction (13 ng/mL), making it
unlikely that a drug interaction on the level of the gut via BCRP inhibition or CYP3A4
induction will occur. This assessment was followed-up and confirmed by a clinical study
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investigating the effect of a 1600 µg oral dose of selexipag on the pharmacokinetics of the
sensitive CYP3A4 substrate midazolam that has a significant gut extraction (Juif PE et al.,
2017, submitted for publication). This study showed that exposure to midazolam and its
metabolite 1-hydroxymidazolam was not affected by co-administration of 1600 g selexipag.
In conclusion, the metabolism pathways of selexipag have been characterized in man and it
has been shown that selexipag has a low potential to inhibit or induce cytochrome P450 and
transporters.
Accepted Manuscript
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Accepted Manuscript
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Figure 1 Chemical structures of [
14
C]selexipag and [
14
C]ACT-333679. Asterisks denote
the position of the radiolabel, which is uniformly distributed throughout both phenyl
rings.
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Figure 2 Identification of metabolites in faecal samples
Panel A: Radiochromatogram of a representative faecal sample after oral administration of [
14
C]selexipag (400
µg/45 µCi) to healthy volunteers. Retention times: P18 (7.5 min), P10 (13.8 min), ACT-333679 (19.4 min),
selexipag (21.9 min)
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Panels B / C: 11 Product-ion mass spectrum of the authentic standard for P10 (B) and
metabolite P10 (C) at m/z 436, (M + H)
+
.
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Panels D / E: 11 Product-ion mass spectrum of the authentic standard for P18 (D) and
metabolite P18 (E) at m/z 436, (M + H)
+
.
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Figure 3 Effect of esterase inhibitors on the formation rate of ACT-333679 from
selexipag in human liver microsomes in the absence of NADPH
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Figure 4 Metabolic profiles of [
14
C]selexipag and [
14
C]ACT-333679 in human liver
microsomes
Panel A: [
14
C]selexipag with human liver microsomes
Panel B: [
14
C]ACT-333679 with human liver microsomes
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Figure 5 Metabolic profile of [
14
C]selexipag and/or [
14
C]ACT-333679 with recombinant
CYP2C8 and CYP3A4
Panel A: [
14
C]selexipag with recombinant CYP2C8
Panel B: [
14
C]ACT-333679 with CYP2C8
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Panel C: [
14
C]selexipag with recombinant CYP3A4
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Figure 6 Metabolic profile of [
14
C]ACT-333679 with recombinant UGT1A3 and
UGT2B7
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Figure 7 Proposed metabolic pathways of selexipag in human
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Table 1 Prevalence of selexipag and its metabolites in human
plasma
Compound
AUC
0-last
1
(pmol/mL*h)
Relative
AUC
2
(%)
Determined
mass
Exact theoretical
mass [M+H]
Change in elemental
composition
Selexipag
38.3
14
497.2221
497.2223
-
ACT-333679
213
78
420.2285
420.2287
- CH3NOS (77)
P11
2.9
0.6
596.2626
596.2608
- CH3NOS (77) +
C6H8O6 (glucuronide,
+ 176)
P10
2.6
0.6
436.2232
436.2236
- CH3NS (61)
P12
12
2.6
378.1817
378.1818
- C3H6 - CH3NS (119)
P14
2.2
0.5
248.1186
248.1188
- C3H6 - C7H13NO4S
P13
7.1
2.6
436.2234
436.2236
- CH3NS (61)
P4
2.6
0.9
436.2235
436.2236
- CH3NS (61)
P34
0.3
0.1
408.2287
408.2287
- C2H3NOS (89)
Pharmacokinetic parameters were estimated using Phoenix™ WinNonlin (version 6, Pharsight Corporation, Mountain View, USA). A
non-compartmental PK analysis was performed calculating AUC
0-last
. Results are expressed in pmol/mL*h in order to account for the
differences in molecular weight.
1
AUC
0-last
were calculated based on the concentrations in acidified plasma,
2
sum of selexipag and all
metabolites = 100%. P35 was present in trace amounts. Full scan LC-MS runs of the sample containing the metabolites and control
sample were processed using the background subtraction tool of Metworks 1.2
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Table 2 Prevalence of selexipag and its main metabolites in vitro in human liver
preparations following incubations with [
14
C]selexipag or [
14
C]ACT-333679
Human hepatocytes
1
Human liver microsomes
2
6.9
73 / 87
3
57
14 / 3.5
3
2.0
6.9 / 5.7
3
16
1.2 /-
3
2.0
2.3 / 1.9
3
-
0.9
-
0.6
-
1.6
Human hepatocytes
Human liver microsomes
57
79
19
16
n.a.
2.7
5.3
2.6
2.1
-
2.4
-
Incubation time:
1
24 h and
2
1 h.
3
Incubation in presence of the carboxylesterase inhibitor BNPP. Numbers are expressed as percent
of total radioactivity in the respective chromatograms; -: absence of a metabolite in the incubation. n.a., data not available.
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Table 3 K
i
values of selexipag and ACT-333679 on several human cytochrome P450 enzymes
K
i
[µM]
Selexipag
ACT-333679
total
free
1
total
free
1
CYP2C8
2.0
0.6
11
7.7
CYP2C9
3.5
1.1
15
10.5
K
i
= inhibition constant;
1
Free K
i
was obtained using the free fraction determined in human liver microsomes, i.e.
30% for selexipag and 70% for ACT-333679 (both, SD < 5%).
Table 4 Regulation of CYP3A4, CYP2C9, and CYP2B6 mRNA in human hepatocytes
EC
50
, mean
Selexipag
ACT-333679
total [µM]
free [nM]
1
total [µM]
free [nM]
1
CYP3A4
2
3.0 ± 0.6
27
8.4 ± 0.6
100
CYP2C9
3
0.8 ± 0.2
7
2.6 ± 0.5
30
CYP2B6
3
9.7 ± 0.3
140
10 ± 1.4
130
EC
50
= concentration that induces 50% of the maximal response;
1
free EC
50
calculated using the fractions
determined in the different media used for the induction experiments, i.e. 0.8-1 % (SD < 5%) for selexipag and 1.1-
1.3% (SD < 5%) for ACT-333679.
2
Mean EC
50
values result from experiments in three independent hepatocyte
batches;
3
Mean EC
50
values result from one experiment in one batch.
Table 5 Inhibition of human transporter proteins by selexipag and ACT-333679
IC
50
, [µM]
selexipag
ACT-333679
total
free
1
total
free
1
OATP1B1
2.4 ± 0.2
1.7
3.5 ± 0.3
2.6
OATP1B3
1.7 ± 0.2
1.2
4.1 ± 0.8
3.1
BCRP
1.9 ± 0.3
0.8
5.6 ± 0.9
2.2
OAT1
1.4 ± 0.2
1.0
25 ± 0.4
20
OAT3
1.7 ± 0.3
1.2
2.1 ± 0.6
1.7
IC
50
= concentration that induces 50% of the maximal response. Mean values are given ± standard deviation (SD).
For P-gp/MDR1, OCT1/2, MATE1, MATE2K, MRP2 and BSEP IC
50
>10 µM.
1
Free IC
50
were calculated using
the mean free fractions determined for selexipag and ACT-333679 in cellular assays, i.e. 70% and 80%,
respectively; free fractions were 40% for both compounds in vesicles. SD on all mean binding values are below 8
%.
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Table 6 Calculation of gut concentration and drug interaction potential of selexipag
BCRP
CYP3A4 induction
fraction absorbed
1
1
absorption rate constant, ka (/min)
1
0.015
0.015
Qent (mL/min)
300
300
I,gut = Fa ka dose/Qent (g/mL)
2
0.081
0.081
1
ka (geometric mean) was determined by 1-compartamental PK modeling of the observed clinical data after oral
selexipag dosing (Kaufmann et al., 2017);
2
using a dose of 1600 g and Qent of 300 mL/min
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